Collagen material and method for producing collagen material

ABSTRACT

A collagen material is characterized in being constituted of a collagen gel fragment wherein a shape of the collagen gel fragment is a string and the string collagen gel fragments couple with each other by drying, and the collagen gel fragments have orientation obtained by giving a flow of a fixed direction to collagen solution during a process of gelating a collagen solution. A method of producing a collagen material comprises a step for preparing a string collagen gel formed and orientated by collagen solution being extruded through a nozzle into a container containing a phosphate buffered saline, and giving a flow of a fixed direction to the collagen solution wherein a shape of the collagen gel is a string and the direction of the orientation of the string collagen gel is a direction of the string shape, a step for obtaining multiple string collagen gels wherein the orientation are controlled, a step for arranging the multiple collagen gels in a desired shape, and a step for drying the multiple collagen gels arranged in the desired shape to couple with the collagen gels each other.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S. patentapplication Ser. No. 14/000,397 filed Aug. 19, 2013, which claimspriority from PCT Application No. PCT/JP2012/001098 filed February 20,2012 and Japanese Patent Application No. JP 2011-035239 filed Feb. 21,2011, which are each expressly incorporated by reference herein.

Technical Field

The present invention relates to a collagen material, and a method ofproducing the collagen material, in particular, a collagen material anda method of producing the collagen material, using a collagen segment.

Background

In the past, a non orientated collagen was used as a substrate materialof a cell culture for many years. On the contrary, a collagen having anorientation is found in tremendous numbers in the human body accordingto the site, and therefore, it is extremely important to design andproduce a shape and a direction of the orientation of the collagenmaterial in a field of regeneration medicine etc., without anyinhibition.

As a method of a collagen substrate having an orientation, it isgenerally known a method of applying a powerful magnetic field to itduring a process of formation of a collagen fiber (“Patent Literature1”). Further, it is also known a method of spin-coating a collagen gel(“Patent Literature 2”).

PRIOR LITERATURE Patent literature

-   Patent Literature 1: JP-A-2006-280222-   Patent Literature 2: JP-A-2010-148691

DISCLOSURE OF THE INVENTION Problems to be Resolved by the Invention

However, in a method of producing a collagen material as described inthe above Patent Literatures 1 and 2, it was impossible to allow aplanar shape or a three-dimensional shape such as a ribbon, a sheet, atube, a block to be freely set, and allow direction of orientation inthe material to be freely set.

Moreover, although there is a technique for orientation of a collagen,there are no technique for designing a shape of collagen material and adirection of orientation in the material freely. As a result of this,there are only non orientated and an orientated collagen material havinga limited shape and a limited direction of orientation.

Therefore, it is an object of the present invention to provide a methodof producing an oriented collagen material which makes it possible todesign a shape and a direction of orientation freely, and an orientedmaterial obtained by the method.

Means of Solving the Problems

In order to accomplish the above object, the present inventors madestrenuous studies as to a collagen material using both a collagen havingorientation and non orientated collagen.

That is, a collagen material according to the present invention, ischaracterized in that the material comprises a collagen gel fragment.

Furthermore, in a preferred embodiment of the above mentioned collagenmaterial according to the present invention, the material ischaracterized in that the collagen gel fragment has orientation.

Furthermore, in a preferred embodiment of the above mentioned collagenmaterial according to the present invention, the material ischaracterized in that the collagen gel fragment has no orientation.

Furthermore, in a preferred embodiment of the above mentioned collagenmaterial according to the present invention, the material ischaracterized in that the collagen gel fragment comprises both thecollagen gel fragment having orientation and the collagen gel fragmenthaving no orientation.

Furthermore, in a preferred embodiment of the above mentioned collagenmaterial according to the present invention, the material ischaracterized in that a shape of the collagen gel fragment comprises atleast one selected from the group comprising a string, a ribbon, asheet, a sponge, a grain, a rod, a ring, a spiral, a spring, a disk, adome or a block.

Furthermore, in a preferred embodiment of the above mentioned collagenmaterial according to the present invention, the material ischaracterized in that the orientation is an uniaxial orientation, aspiral orientation, a double axis orientation, a two-dimensionalorientation, a triaxial orientation, or a three-dimensional orientation.

Furthermore, in a preferred embodiment of the above mentioned collagenmaterial according to the present invention, the material ischaracterized in that a part or all of the collagen gel fragment iscoated on a substrate comprising metal, ceramics, high-polymer materialor biomaterial.

Furthermore, in a preferred embodiment of the above mentioned collagenmaterial according to the present invention, the material ischaracterized in that the collagen gel fragment includes a cell growthpromotant.

Furthermore, in a preferred embodiment of the above mentioned collagenmaterial according to the present invention, the material ischaracterized in that the cell growth promotant is Epidermal growthfactor (EGF), Insulin-like growth factor (IGF), Transforming growthfactor (TGF), Nerve growth factor (NGF), Brain-derived neurotrophicfactor (BDNF), Vesicular endothelial growth factor (VEGF),Granulocyte-colony stimulating factor (G-CSF),Granulocyte-macrophage-colony stimulating factor (GM-CSF),Platelet-derived growth factor (PDGF), Erythropoietin (EPO),Thrombopoietin (TPO), basic fibroblast growth factor (bFGF or FGF2), orHepatocyte growth factor (HGF).

Furthermore, in a preferred embodiment of the above mentioned collagenmaterial according to the present invention, the material ischaracterized in that a shape of the material is a ribbon, a sheet, atube, a sponge, a grain, a rod, a ring, a spiral, a spring, a disk, adome or a block.

Furthermore, in a preferred embodiment of the above mentioned collagenmaterial according to the present invention, the material ischaracterized in that the material is designed so as to become almostequal to the orientation of each site of a normal tissue from a livingorganism.

Furthermore, in a preferred embodiment of the above mentioned collagenmaterial according to the present invention, the material ischaracterized in that the material contains oxygen.

Furthermore, a method of producing a collagen material according to thepresent invention, a method is characterized in that a method comprises;

-   -   a step for preparing a collagen gel fragment,    -   a step for arranging the collagen gel fragments in a desired        shape, and a step for drying the collagen gel fragments arranged        in the desired shape.

Furthermore, in a preferred embodiment of the above mentioned method ofproducing a collagen material according to the present invention, amethod is characterized in that a method further comprises;

-   -   a step for imparting an orientation to the collagen gel        fragments.

Furthermore, in a preferred embodiment of the above mentioned method ofproducing a collagen material according to the present invention, amethod is characterized in that the desired shape is planar shape and/orthree-dimensional shape.

Furthermore, in a preferred embodiment of the above mentioned method ofproducing a collagen material according to the present invention, amethod is characterized in that the desired shape is a ribbon, a sheet,a tube, a sponge, a grain, a rod, a ring, a spiral, a spring, a disk, adome or a block.

Furthermore, in a preferred embodiment of the above mentioned method ofproducing a collagen material according to the present invention, amethod is characterized in that the step for drying the collagen gelfragments is carried out by freeze dry.

Furthermore, in a preferred embodiment of the above mentioned method ofa collagen material according to the present invention, a method ischaracterized in that the method further comprises;

-   -   a step for introducing bubble into the collagen gel fragment, by        means of any one method of a pressure-reduced pressure method, a        gas-liquid shearing method and/or a method of introducing bubble        using a membrane having a pore.

Moreover, a collagen material according to the present invention ischaracterized in that the material is obtained by the method ofproducing a collagen material according to the above present invention.

Effect of Invention

The present invention has an advantage effect that it is possible todesign a shape and a direction of orientation as to an orientatedcollagen material freely. Further, the present invention has anadvantage effect that it is possible to produce a biocompatible materialwhich an orientation is controlled so that the orientation can be nearlyequal to an orientation of each site of a normal tissue of a livingorganism, and further to put into practice regeneration of normal tissueof a living organism, since it is possible to produce a material havingan orientation of collagen according to a site in a tissue from a livingorganism.

The present invention has an advantage effect that it is possible toproduce a collagen material at a macro size of a millimeter order ormore. Moreover, the method of producing a collagen material according tothe present invention has an advantage that it is easy to manufactureand it is possible to produce a collagen material capable of beingdesigned their shape freely. Further, the present invention has anadvantage effect that if an orientated collagen with a string shape isaligned to a desired direction in a plane shape or a three-dimensionalshape, it is possible to design a direction of orientation in thecollagen material freely.

Furthermore, the present invention has an advantage effect that even ifa dried orientated or non orientated collagen material is immersed intoPBS or cell culture medium etc, or is implanted into the living body,the shape of the material is maintained for necessary period and is notdissolved to the original shape of the collagen gel fragment such as astring shape etc. The reasons why the shape of the material ismaintained for necessary period and it is difficult to dissolve, isassumed that according to a triple-strand helix conformation which isone of feature of collagen molecule, a hydrophobic residue is throwedout to outside to make their residues assemble and be firmly fixed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 gives a photograph of an orientated collagen gel with a stringshape immediately after it is produced in PBS.

FIG. 2 gives a photograph of a dried orientated collagen material (FIG.2(b))with a ribbon shape obtained by drying after an orientated collagengel with a string shape is aligned to a ribbon shape

(FIG. 2(a). An orientation of the ribbon shape in inside of sample is inparallel with a travel direction of an orientated collagen with a stringshape.

FIG. 3 gives a photograph in the case that an orientated collagen gelwith a string shape is aligned to two layers (FIG. 3(a)), and a firstlayer and a second layer are perpendicular as to a travel direction ofan orientated collagen with a string shape. FIG. 3(b) shows those thatan orientated collagen gel with a string shape is aligned and after thatbeing dried, and FIG. 3(c) shows those of the end of the driedorientated collagen gel being cut.

FIG. 4 gives a photograph of a dried orientated collagen material with atube shape made by obtaining a sheet shape using an orientated collagengel with a string shape, and after that, winding the sheet for an axlerod, and removing the axle rod from the sheet.

FIG. 5 gives a raman spectrum of an orientated collagen, (i) shows aspectrum in the case that a direction of a laser polarization is inparallel with a travel direction of a collagen, (ii) shows a spectrum inthe case that a direction of a laser polarization is perpendicular to atravel direction of a collagen, respectively.

FIG. 6 gives an example of a dome type of the collagen material. FIG. 7gives a result that an orientation test is performed as to theorientated sheet and non orientated sheet.

Mode for Carrying Out the Invention

A collagen material according to the present invention comprises acollagen gel fragment. That is, a collagen material of the presentinvention is a collagen material comprising one or more collagen gelfragment(s). In the past, in the case that a collagen material isprepared, a powdered state of collagen is dissolved in solution toprepare collagen solution, the collagen solution is used as a statesubstance, and the collagen solution is gelated by a gelatinizing agentetc., to obtain a desired sheet. On the other hand, in the presentinvention, it is possible to obtain a collagen material by preparing acollagen gel fragment as a start substance, and then coupling eachcollagen gel fragments as mentioned above. The coupling of each collagengel fragments may be carried out by using a gelatinizing agent etc.Further, as one of the feature of the present invention, it is possibleto carry out the coupling of each collagen gel fragment without using aspecific gelatinizing agent. That is, it is possible to align thecollagen gel fragment in a desired shape, and to dry and fix it tocouple each collagen gel fragments.

A collagen is included in every tissue in the body such as skin, muscle,internal organ, bone. A collagen is different from other protein, andmakes structure made by fiber or membrane in a space between a cell anda cell, that is, in outside of a cell. Almost collagen exists as aninsolubility substance. That is, it is thought that in the living body,a collagen plays a role in something like a binder capable of coupling acell and a cell, and as well as plays a role in a barrier for aligning acell in a correct position. That is, a collagen makes it possible tomake, serve as a backbone of and couple the conformation of every partof the body, internal organ etc., to make borderline between a cell. Thecollagen is so called as a cell matrix.

A collagen molecule has about 300 nanometer of length, about 1.5nanometer in diameter, and a shape of a rod, and the molecule has atriple-strand helix structure (helix structure) wherein three chainstwist together with each other. The collagen molecule is coupled witheach other with connecting bridges thereof to increase the intensity ofcoupling. As a feature of the collagen molecule, it has a property thatit will be solved by heat, this is an opposite feature comparing with ageneral protein having a feature that it will be solidified by heat. Ingeneral, so called gelatinization means that three chains come loose todivide into a single strand. A temperature capable of generating thechange is generally called as a denaturation temperature, it is about40° C. or more in a collagen of the land-dwelling creature, it is about0 to 25° C. in a collagen of the heterothermic animal.

In the present invention, it is possible to obtain a collagen materialwherein a collagen gel fragments are coupled with each other. Althoughdetails of their mechanism is unclear, since it is possible to set in adesired shape and to be solidified by drying, it is thought that theregeneration of each collagen gel fragment makes it possible to coupleand stabilize them without using a gelatinizing agent. Moreover, a stepfor drying may be general natural seasoning or freeze drying.

Furthermore, in a preferred embodiment of the above mentioned collagenmaterial according to the present invention, the collagen gel fragmenthas orientation. At this moment, a simple explanation as to theorientation is as follows.

First of all, an explanation as to a collagen having an orientation isas follows. A collagen having an orientation means a collagen which atravel direction of a fibrous collagen such as a single collagen gel ora dry collagen gel uniforms in some direction. In the case that acollagen having an orientation is coated in a substrate comprising ametal, a ceramics, a high-polymer material or a biomaterial (hereinafterit is a collagen substrate), a collagen having an orientation means acollagen which a travel direction of a fibrous collagen such as a singlecollagen gel or a dry collagen gel coated on a substrates such as ametal, a ceramics, a high-polymer material or a biomaterial formed tovarious shapes, uniforms in some direction.

An advantage of the use of a collagen gel having an orientation as astarting material is as follows. That is, the reasons are that the useof a collagen having an orientation in a curve within a collagen as astating material makes it possible to also produce an orientation in acurve in an osteoblast. Further, in the present invention, it isessentially possible to develop an osteoblast with an orientation at a“surface” of collagen (substrate), although it is also thought that anosteoblast gets into inside of a collagen (substrate), it is possible toinclude such cases in the present invention. Furthermore, according tothe present invention, if an orientated collagen gel having some shapeand some direction of an orientation as a starting material is prepareddepending on a needs of an orientated material as an end product, it ispossible to produce an orientated collagen material with a larger sizethan a millimeter order by controlling a shape or a direction of anorientation without any inhibition.

A method of preparing a collagen gel having an orientation is notparticularly limited, but according to a common procedure. For example,as a method of imparting an orientation to a collagen gel at a largersize than a millimeter order, a method of giving a flow of a fixeddirection to collagen solution during a process of gelating a collagensolution is suggested, but other method may be used. As other method,mention may be made of a method of applying a strong magnetic fieldduring a process of forming a collagen fiber, a method of spin-coating acollagen gel, a method of drawing a collagen gel to a predetermineddirection mechanically (and physically).

In the case that a collagen gel fragment having an orientation isprepared by a method of applying a strong magnetic field during aprocess of forming a collagen fiber, since a collagen fiber is set inarray in a perpendicular direction to the magnetic field, if it is keptto apply a magnetic field from the same direction, it is possible toobtain a two-dimensional orientation, and if it is kept to apply arotational magnetic field, it is possible to obtain a single axisorientation. It is possible to use a method of applying a magnetic fieldif such collagen gel having an orientation is used as a startingmaterial. However, if the magnetic field is used, basically, it ispossible to produce only those of the collagen having a uniformorientation, and a macro shape also tends to be limited. On the otherhand, in the case that a collagen gel having an orientation is preparedby a method of giving a flow of a fixed direction to collagen solutionduring a process of gelating a collagen solution. it is possible toproduce a collagen having a different 3D orientation by forming andlaminating a various shape including a sheet-like shape because of theuse of a flow of liquid.

In such method, an orientated collagen (single collagen) in the examplecan be obtained by using a flow of a collagen solution to give anorientation to a collagen during a process of obtaining a collagen gel.

Although it is a string-shaped of a collagen in the photograph of theexample, it is possible to produce an orientated collagen gel or acollagen gel fragment with various shapes (line, plane, three dimension)such as a ribbon-shaped with a large width. Further, during a process, acontrol of a velocity of a flow also makes it possible to control adegree of the orientation. Therefore, since it is possible to control adirection of the orientation or a degree of the orientation therebygiving a desired distribution even if it is in the same collagen gel,the use of such collagen gel or collagen gel substrate according to thepresent invention also makes it possible to control a direction of theorientation and a degree of the orientation (that is control ofdistribution of the orientation). Moreover, controlling of theorientation manly includes two meaning: At first, (1) it is possible tomake an orientation to the collagen material itself freely, and (2) itis possible to control a growth of a cell and a tissue on the line withan orientation of the collagen material in the case that a cell iscultured by using the collagen material having those orientation, or inthe case that a living tissue is regenerated. In the present invention,it is possible to control these two orientations as mentioned above.

For example, an explanation as to a method of giving a flow of a fixeddirection to collagen solution during a process of gelating a collagensolution is as follows. Although a concentration of a collagen solutionis preferably 10 mg/mL or more from a viewpoint that an obtainedcollagen or a collagen substrate can have an enough mechanical strength,it may be about 3 mg/mL or more. An origin of a collagen is not limited.Further, a seed, a site of a tissue, an age etc., of an animal derivedfrom are not particularly limited. For example, it is possible to useone derived from animals such as a rat tail, a pig hide, a cowskin, acamel bird, a fish. That is, it is possible to use a collagen obtainedfrom a skin, a bone, a cartilage, a tendon, an internal organ of amammal (for example, such as a cattle, a pig, a horse, a rabbit, a mouseetc.) or birds (for example, chicken etc.). A collagen like proteinderived from a skin, a bone, a cartilage, a fin, a fish scale, aninternal organ of fishes (for example, such as a pacific cod, aparalichthys olivaceus, a flatfish, a salmon, a trout, a tuna, a chubmackerel, a sea bream, a sardine, a shark etc.) may be used. Moreover, amethod of extracting a collagen is not particularly limited, but acommon method of extracting may be used. Further, a collagen obtained bya recombinant DNA technique other than that from the extraction from ananimal tissue, may be used. Further, in order to suppress anantigenecity, an enzyme-treated atelocollagen may be used. Further, as acollagen, an unmodified soluble collagen such as an acid solublecollagen, a salt soluble collagen, an enzyme soluble collagen(atelocollagen), a chemically-modified collagen such as acylation suchas succinylation, phthalation, esterification such as methylation,deamination of alkali solubilization, and further an insoluble collagensuch as a tendon collagen etc., may be used. Further, a chemicalcross-linking agent, a medicinal agent or an air bubble such as oxygenmay be introduced into a collagen solution. A method of introducing themis not particularly limited, but according to a common procedure.

It is possible to quantitatively assess a direction of orientation or adegree of orientation as to an obtained collagen by using, for example,a raman spectroscopy microscope. A raman spectroscopy is to examine acomponent which a frequency modulation of a scattered light caused byhitting against molecular is occurred according to molecular vibration,by means of the use of a spectroscope, and thereby making it possible toobtain information as to composition of a target for analysis or acrystal structure to analysis an orientation of collagen.

Furthermore, in a preferred embodiment of the above mentioned collagenmaterial according to the present invention, the collagen gel fragmenthas no orientation. Because even if the collagen gel fragment having noorientation is used as a stating material, it is possible to obtain acollagen material by means of aligning the collagen gel fragment in adesired shape to dry and be firmly fixed. Furthermore, in a preferredembodiment of the above mentioned collagen material according to thepresent invention, is characterized in that the collagen gel fragmentcomprises both the collagen gel fragment having orientation and thecollagen gel fragment having no orientation. A method of producing thecollagen gel fragment having no orientation is not particularly limited,but according to a common procedure. It is possible to obtain thecollagen gel fragment having no orientation if the step for giving anorientation is omitted in the above method of producing the collagen gelfragment having orientation.

Moreover, in the above, although the collagen gel fragment with a stringshape is mainly explained, in a preferred embodiment of the abovementioned collagen material according to the present invention, a shapeof the collagen gel fragment may comprise at least one selected from thegroup comprising a string, a ribbon, a sheet, a sponge, a grain, a rod,a ring, a spiral, a spring, a disk, a dome or a block.

Furthermore, in the case that it will be explained from a viewpoint ofthe control of orientation, it is possible to control a direction oforientation and a degree of orientation of the collagen material bymeans of controlling a direction of orientation and a degree oforientation of the collagen gel or the collagen gel fragment.

Furthermore, in a preferred embodiment of the above mentioned collagenmaterial according to the present invention, is characterized in thatthe orientation is an uniaxial orientation, a spiral orientation, adouble axis orientation, a two-dimensional orientation, a triaxialorientation, or a three-dimensional orientation.

Furthermore, in a preferred embodiment of the above mentioned collagenmaterial according to the present invention, a part or all of thecollagen gel fragment is coated on a substrate comprising metal,ceramics, high-polymer material or biomaterial. Moreover, a method ofcoating is not particularly limited, but according to a commonprocedure. Further, in the present invention, it is one of the futurethat the above collagen material is a macro size at a larger size than amillimeter order.

Furthermore, in a preferred embodiment, from a view point that a cellgrow up for a short period to accelerate the regeneration of tissue, thecollagen gel fragment includes a cell growth promotant. As the cellgrowth promotant, mention may be made of Epidermal growth factor (EGF),Insulin-like growth factor (IGF), Transforming growth factor (TGF),Nerve growth factor (NGF), Brain-derived neurotrophic factor (BDNF),Vesicular endothelial growth factor (VEGF), Granulocyte-colonystimulating factor (G-CSF), Granulocyte-macrophage-colony stimulatingfactor (GM-CSF), Platelet-derived growth factor (PDGF), Erythropoietin(EPO), Thrombopoietin (TPO), basic fibroblast growth factor (bFGF orFGF2), or Hepatocyte growth factor (HGF), or one or more combinationthereof.

Furthermore, in a preferred embodiment of the above mentioned collagenmaterial according to the present invention, a shape of the material isa ribbon, a sheet, a tube, a sponge, a grain, a rod, a ring, a spiral, aspring, a disk, a dome or a block. These can be prepared by a manner oforientation of the above collagen gel fragment, or a process afteraligning it to dry and be firmly fixed. It is possible to carried out asecondary process as to the collagen material consisting of the collagengel fragment of the present invention. That is, it is possible toprepare a sheet type of collagen material (fragment) from a string shapeas shown in FIG. 1 and to further process the collagen material to makea many sort of a final shape type of the three dimensional collagenmaterial.

Furthermore, in a preferred embodiment, the material is characterized bybeing designed so as to become almost equal to the orientation of eachsite of a normal tissue from a living organism. The collagen materialdesigned so as to become almost equal to the orientation of each site ofa normal tissue from a living organism can be used as a material for theregeneration medicine without modification.

In the present invention, it is possible to basically produce a dry typeof the collagen material. However, it is also possible to produce geltype of the collagen material, for example, obtained by immersing a drytype of the collagen material into PBS etc. In general, although a partof tissue of the collagen material may be destroyed if it is dried, thedry type of material will be easy to use from a view point of storagestability (it is easy to maintain a shape. A gel is easy to corruptsince it contains water.), transit performance (A gel is easy to destroysince it contains water. It may become deformed when it is took up froma vessel.)

In the present invention, a dry type of the collagen material can beused as a gel type of the collagen material by setting back it to a gelwith PBS, medium when using it actually. In the present invention, a drytype of the collagen material become increased in density of collagenfiber tissue by drying to drop out of water from a gel (It contains 90%or more of water.). Even if it is set back to a gel with PBS or mediumagain, it will be a small size in volume comparing with an originaldried collagen material. As a result of this, the dry type of thecollagen material has a lot of advantage, such as strength ororientation comparing with those of a gel type when manufactured, sincean increase in density of the tissue remains.

In such manner, as one of a feature in the present invention, it ispossible to produce a dry type of the collagen material as well as a geltype of the collagen material by setting back it to a gel with PBS ormedium.

Furthermore, in a preferred embodiment of the above mentioned collagenmaterial according to the present invention, the material ischaracterized by containing oxygen. In the collagen material, anadvantage of containing oxygen is as follows. That is, in the case thatthe collagen material contains oxygen, it is though that there are a fewmerit, such as (a) it become a resource of air (oxygen) to a cell in thecase that a cell grows up in inside of the collagen material, andfurther (b) it gives a space making it possible to grow up a cell intoinside of the collagen material.

Although oxygen may include bubble, an embodiment of containing oxygenwill be explained as follows. That is, containing of oxygen may benearly equal to containing bubble. As the component, (1) in the casethat it contains air (which is general air containing oxygen, nitrogen,carbon dioxide), (2) in the case that a low concentration or a highconcentration of oxygen is contained in air (which also includes thoseof the content of the other components such as nitrogen or carbondioxide being changed depending on increase and decrease of oxygenconcentration.), (3) in the case that it contains only oxygen. It isthough that there are at least the above three embodiments of containingoxygen.

A method which once a collagen gel fragment is dried may be a generalmethod for using since there is a possibility that a collagen gelfragment is difficult to couple each multiple collagen gel fragment andthereby is difficult to become a stable and complicated shape of acollagen material, and easy to separate these fragments.

If the collagen is dried, it is a fiber. However, a collagen sheet has afeature which is not easy to pass water. Therefore, all of theintroduced bubble is not blowed up as gas. A part of at least oxygen andbubble remains to be trapped in inside of the collagen material even ifit is dried.

If a bubble is introduced into a collagen solution and a content of thecollagen solution increases from 100 to 110, 10 of a ration of thebubble will be remained. However, it is though that some part of abubble is blowed up during a dry process of the collagen material.Although if the collagen gel is not dried, almost all contents of thebubble which is introduced into the collagen solution is fixed in thecollagen gel, the contents of the bubble which is blowed up, and whichremains in the gel during a dry process will be case by case. if thereis 100 of the collagen solution and 1% of the concentration of collagen,99% is water. It is thought that some extent of bubble will be blowed uptogether with the 99% of water.

Next, a method of producing the collagen material of the presentinvention will be explained. The method of producing the collagenmaterial of the present invention comprises;

-   -   a step for preparing a collagen gel fragment,

a step for arranging the collagen gel fragments in a desired shape, anda step for drying the collagen gel fragments arranged in the desiredshape. A step for drying the collagen gel fragments may be include bothgeneral natural seasoning and freeze dry. An explanation as to acollagen gel fragment can be directly applied to and referred to theexample using the collagen gel fragment in the explanation of the abovecollagen material according to the present invention. It is possible toalign a collagen gel fragment in the line with a shape etc., of a finalproduct collagen material, and dry the collagen gel fragment and therebycoupling the collagen gel fragment each other, and finally obtaining thecollagen material consisting of the collagen gel fragment. Moreover, itis difficult to check a boundary of the collagen gel fragment with eyesafter the collagen gel fragments are dried and coupled each other.

Furthermore, in a preferred embodiment, the step for drying the collagengel fragments may be carried out by freeze dry. Although there are theabove merits in the case of a natural seasoning, by contrary, it isthought that the volume of the fragment become reduced in size comparingwith the original fragment, and thereby getting smaller a space forgrowing up a cell into an inside of the collagen material. It ispossible to avoid these problems in the case of freeze dry. That is, inthe case of freeze dry, it is possible to produce a collagen gelfragment and after that, to fixed with each fragments and maintain acondition that the inside of the fragment, as a result, the inside ofall the produced fragment become hollowed out. By doing this, it is easyto grow up a cell into inside of the collagen material.

Furthermore, in a preferred embodiment, is characterized in that themethod further comprises;

-   -   a step for arranging an orientation to the collagen gel        fragment.

A method for preparing an orientation to the collagen gel fragment isnot particularly limited, but according to a common procedure. Forexample, it is possible to apply to the above method of producing theorientated collagen gel fragment, in the case of giving an orientationto the collagen gel fragment. That is, mention may be made of a methodof using the above collagen solution, a method of using a magneticfield, and a method of spin-coating a collagen gel, and it is notparticularly limited.

Furthermore, in a preferred embodiment, a method is characterized inthat the desired shape is planar shape and/or three-dimensional shape.Specifically, as the desired shape, mention may be made of a ribbon, asheet, a tube, a sponge, a grain, a rod, a ring, a spiral, a spring, adisk, a dome or a block. These shapes can be obtained by secondaryprocess or tertiary process etc., of the collagen gel fragment.

Furthermore, in a preferred embodiment of the method of producing thecollagen material according to the present invention, is characterizedin that the method further comprises;

-   -   a step for introducing bubble into the collagen gel fragment, by        means of any one method of a pressure-reduced pressure method, a        gas-liquid shearing method and/or a method of introducing bubble        using a membrane having a pore. At this moment, “introducing        bubble” is for introducing oxygen. That is, it is for obtaining        a collagen material containing oxygen. A merit of containing        oxygen may be applied to and referred to the above explanation        of the collagen material containing oxygen.

Although oxygen may include bubble, an embodiment of containing oxygenwill be explained as follows. That is, containing of oxygen may benearly equal to containing bubble. As the component, (1) in the casethat it contains air (which is general air containing oxygen, nitrogen,carbon dioxide), (2) in the case that a low concentration or a highconcentration of oxygen is contained in air (which also includes thoseof the content of the other components such as nitrogen or carbondioxide being changed depending on increase and decrease of oxygenconcentration.), (3) in the case that it contains only oxygen. It isthough that there are at least the above three embodiments of containingoxygen.

in the similar way of the case of the addition of “cell growthpromotant”, it is for obtaining a condition that “bubble (the above air,or oxygen etc.)” is contained in the collagen gel fragment. Further,specifically, it is preferable to introduce bubble at a stage ofpreparing the collagen gen fragment, as these method, the followingmethod can be explained with an example.

For example, a proportion of air is introduced into a vessel with acover which a collagen solution is added, and is mechanically produced avibration in the vessel after covered, and is gelated the collagensolution containing bubble. There is a possibility that a sufficientsmall bubble can not be obtained if air is just mixed into the collagensolution by producing a vibration in the vessel. Moreover, in the casethat a small bubble such as so called nano bubble is needed, thefollowing methods can be used.

For example, it is possible to introduce a smaller bubble than those ofbeing made by shaking with hand or shaking mechanically, if air is addedinto the collagen solution, and then the solution is shook withultrasonic wave etc.

Moreover, in a preferred embodiment according to the present invention,the method further comprises;

-   -   a step for introducing bubble into the collagen gel fragment, by        means of any one method of a pressure-reduced pressure method, a        gas-liquid shearing method and/or a method of introducing bubble        using a membrane having a pore. At first, in the case of        introducing a micro bubble will be explained. In the case of        introducing a micro bubble, it is possible to introduce the        bubble into the collagen material by using a pressure-reduced        pressure method, or a gas-liquid shearing method. At first, the        pressure-reduced pressure method will be explained. The        pressure-reduced pressure method is a method wherein a large        amount of air is dissolved under the conditions of adding a high        pressure to produce bubble again by reducing the pressure.        Moreover, a gas-liquid shearing method is a method wherein an        eddy-current (400 to 600 revolutions per second) is produced,        and air is sucked in the eddy-current, and the air is cut and        grinded by a fan etc., to generate a lot of bubble. These        methods can be applied to the present invention.

Moreover, it is possible to use a method of introducing bubble using amembrane having a pore, in the case that a smaller size of nano bubblethan micro bubble is needed to introduce the collagen material. Thismethod is a technology of applying pressure on gas and thereby emittingthe gas from a membrane which countless numbers of nano level of hole isopened, to generate a nano bubble. As a membrane, a Shirasu Porous Glass(SPG) membrane is used. It is possible to introduce bubble, microbubble, or nano bubble etc., into the collagen material according tothese common procedure at a desired level.

As mentioned above, the process of the method is simply summarized asfollows. That is, the prepared orientated collagen gel with a stringshape is aligned at a desired shape. The align may be a plane shape or athree-dimensional shape obtained by laminating one or more layer.Moreover, a direction of a string shape in the plane shape or thethree-dimensional shape is a direction of orientation. Therefore, theorientated collagen gel with a string shape can be linearly aligned inthe case that a linearly orientation is needed, the orientated collagengel with a string shape can be roundly aligned in the case that aroundly orientation is needed.

The dryness of the orientated collagen gel with a string shape makes itpossible to firmly fix the collagen gel each other. In this way, acollagen material of the present invention can be obtained by the methodof producing the collagen material according to the above presentinvention. That is, it is possible to obtain the orientated collagenmaterial capable of freely designing a shape and a direction oforientation by means of using the orientated collagen gel with a stringshape, and then aligning it to a plane shape or a three-dimensionalshape such as a ribbon, a sheet, a tube, or a block, and after thatdrying it.

According to the manufacturing method according to the presentinvention, it is also possible to obtain the collagen material capableof freely designing a shape by means of using the collagen gel with astring shape, and then aligning it to a plane shape or athree-dimensional shape such as a ribbon, a sheet, a tube, or a block,and after that drying it, even if the collagen gel with a string shapehas not orientation.

EXAMPLE

At this moment, the present invention will be concretely explained inmore detail with reference to Examples, but the invention is notintended to be interpreted as being limited to Examples. Moreover, it ispossible to change appropriately without departing from the scope of theinvention.

Example 1

In a method of the present invention, first of all, a collagen gel wasprepared as an orientated collagen (substrate) having a size of amillimeter order or more. As to a collagen gel, 9.3 mg/mL concentrationof I type of collagen solution derived from a rat tail (BD Company) wasextruded through a nozzle having 0.38 mm of an inside diameter into aplate container containing a phosphate buffered saline (PBS) at 38° C.,at pH7.4, and thereby sliding the nozzle to obtain a string likecollagen gel having about 1 mm in diameter, about 20 mm of length.

An orientation of a collagen gel obtained thus was analyzed by a ramanspectroscopic microscope (PHOTON Design Corporation). In doing so, anexcitation wavelength was set at 514.5 nm using a continuous oscillationargon ion laser Stabilite 2017 (Spectra-Physics, Inc.), a HR-320 (JovinYvon S.A.S.) as a spectroscope and a LN/CCD-1100-PB/UV AR/1 (Roperscientific Inc.) were used respectively. FIG. 5 gives a result ofevaluating a strength of an amide I and an amide III in the case that adirection of a laser polarization is in parallel with and isperpendicular to a travel direction of a collagen. A peak derived froman amide I depends on a vibration of a C═O bonding located in adirection which is perpendicular to a collagen fiber, and a peak derivedfrom an amide III depends on a vibration of a C—N bonding located in adirection which is in parallel with and perpendicular to a collagenfiber. According to FIG. 5, as to a spectrum wherein a direction of alaser polarization is in parallel with a travel direction of a collagen,compared with a strength of a peak depending on a CH₃ bending vibrationaround 1450 cm⁻¹, a peak strength of amide I (1670 cm⁻¹) as to aperpendicular direction was higher than that of a parallel direction.Further, in a spectrum wherein a direction of a laser polarization isperpendicular to a travel direction of a collagen, a peak of a vibrationdepending on a C—N bonding located in a direction perpendicular to acollagen fiber was observed around raman shift, 1270-1300 cm⁻¹. That is,it was recognized that a collagen fiber was oriented to a long axesdirection of a collagen gel.

Example 2

Next, a larger size of the collagen material is tried to obtain. Firstof all, a orientated collagen gel with a string shape was prepared. Asto a collagen gel, 9.3 mg/mL concentration of I type of collagensolution derived from a rat tail (BD Company) was extruded through anozzle having 0.38 mm of an inside diameter into a plate containercontaining a phosphate buffered saline (PBS) at 38° C., at pH7.4, andthereby sliding the nozzle to obtain a string like collagen gel havingabout 1 mm in diameter, about 200 mm of length. FIG. 1 gives aphotograph of an orientated collagen gel with a string shape immediatelyafter it is produced in PBS.

The produced orientated collagen gel with a string shape is aligned on aplane as shown in FIG. 2, and after that is dried to obtain a driedorientated collagen material with a sheet shape.

Example 3

The above mentioned orientated collagen gel with a string is aligned onthe plane with a sheet shape. After that, a second layer is alignedwherein a direction of an orientated collagen gel with a string shape isperpendicular to a travel direction of the orientated collagen with astring shape of the first layer (FIG. 3(a)). After that, a dried sampleis shown in FIG. 3(b), a sample cut in rectangles after drying is shownin FIG. 3(c). In this way, it is possible to make not only single layerbut multiple layers using the orientated collagen sheet with a sheetshape, and further it is possible to freely design a direction oforientation of each layer.

Example 4

The above mentioned orientated collagen gel with a string shape wasdesigned on a plane to make a sheet shape, and after that, was dried.After that, the obtained sheet is winded for an axle rod, and the axlerod is removed from the sheet to obtain a dried orientated collagenmaterial with a tube shape (FIG. 4). It is possible to freely design adirection of orientation of the collagen material with a tube shapedepending on a direction of winding a sheet having orientation to theaxle rod, since a direction of orientation of the collagen material witha sheet shape is equal to a travel direction of the orientated collagenwith a string shape. That is, it is possible to freely design an angleto a direction such as a direction of a tube axial, a circumferentialdirection of a tube, or from a direction of a tube axial.

Further, it is possible to freely design a diameter of a tube accordingto a size of the axle rod. Further, it is possible to make multiplelayers of the collagen material with a tube shape and to freely design adirection of orientation in each layer of these multiple layers.

Example 5

Next, it was tried to produce a dome type of the collagen material. FIG.6 gives an example photograph of a dome type of the collagen material. Adimension is not measurement, but scale display. Although FIG. 6 gives aphotograph at the time of drying, in order to emphasize a transparency,among four sheet samples in a black back ground of one sheet sample awhite character “C” is set. The four sheets consist of 20 layers whereina direction of orientation is perpendicularly intersected with everylayer. Moreover, in figure, a yellow arrow shows a direction oforientation. In figure, although it is recognized that there are somereflection in the outside of a dome which is looked like a circle, thisshows that the reflecting light can be observed since it is a circle aswell as a dome type. That is, although the above material having 20layers is itself “three-dimensional structure” of the collagen sheet,further, it is recognized that it is possible to make (and dry) not onlya flat disk type using these 20 layers, but also a dome type, andtherefore, it is also possible to make more “complicatedthree-dimensional structure”. This dome type of the sample is aimed tothe regeneration of cornea, and thereby these curvature makingconsistent with a curvature of eyes.

In this way, it is recognized that it is possible to produce a widevarious sort of shape according to the collagen material of the presentinvention, and to make a meaningful contribution to a regenerationmedicine.

Moreover, the orientation of the collagen material with a sheet shapewas evaluated with other measuring method. FIG. 7 gives a result that anorientation test is performed as to the orientated sheet and nonorientated sheet. That is, this is a figure shows that both orientationof dried orientated collagen sheet and dried non orientated collagensheet produced by the above method of producing the collagen material isquantitatively evaluated. Both thicknesses of the sheets was about 10micrometer. This figure shows evaluation of the “phase difference” inthe samples according to an ellipsometry devise using a parallel nicolrotation method. The Retardation in the figure means a phase difference,a phase difference in a rectangular sheet is shown in the contour map.It is recognized that this phase difference in the case of theorientated sheet is higher than that of non orientated sheet, andfurther a direction of the contour map increases in length from side toside (Although it is difficult to recognize, in the orientated sheet, ared-tinged part showing 187.8 to 207.9 nm is observed in several part,while in non orientated sheet these are almost never observed in thesame region. Moreover, in the orientated sheet, a yellow part showing127.5 to 147.6 nm is recognized in a lot of region (about 10 to 20%region of total region), while in non orientated sheet these are onlyobserved in a slight amount of region in several part. In non orientatedsheet, almost region become a blue to a dark green region showing 0 to3.93 nm.). A direction from side to side in figure is a direction oforientation of the orientated collagen, that is, a direction wherein astring having orientation is aligned.

INDUSTRIAL APPLICABILITY

The present inventions make it possible to contribute to a therapy fordisorder and a regenerative medicine and dental fields (in particular,orthopaedic surgery, a cerebral surgery, a medical dentistry) and basicmedical fields. Moreover, it is possible to freely design a collagenmaterial having an orientation according to these site existing in vivoof human, and to contribute to a regenerative medicine and basic medicalfields.

What is claimed is:
 1. A collagen material characterized in beingconstituted of a collagen gel fragment wherein a shape of the collagengel fragment is a string and the string collagen gel fragments couplewith each other by drying, and the collagen gel fragments haveorientation obtained by giving a flow of a fixed direction to collagensolution during a process of gelating a collagen solution.
 2. A collagenmaterial according to claim 1, wherein the material further includes acollagen gel fragment with no orientation.
 3. A collagen materialaccording to claim 1, wherein the collagen gel fragment comprises boththe collagen gel fragment having orientation and the collagen gelfragment having no orientation.
 4. A collagen material according toclaim 1, wherein the orientation is an uniaxial orientation, a spiralorientation, a double axis orientation, a two⋅dimensional orientation, atriaxial orientation, or a three-dimensional orientation.
 5. A collagenmaterial according to claim 1, wherein a part or all of the collagen gelfragment is coated on a substrate comprising metal, ceramics,high-polymer material or biomaterial.
 6. A collagen material accordingto claim 1, wherein the collagen gel fragment includes a cell growthpromotant.
 7. A collagen material according to claim 6, wherein the cellgrowth promotant is Epidermal growth factor (EGF), Insulin-like growthfactor (IGF), Transforming growth factor (TGF), Nerve growth factor(NGF), Brain-derived neurotrophic factor (BDNF), Vesicular endothelialgrowth factor (VEGF), Granulocyte-colony stimulating factor (G-CSF),Granulocyte-macrophage-colony stimulating factor (GM-CSF),Platelet-derived growth factor (PDGF), Erythropoietin (EPO),Thrombopoietin (TPO), basic fibroblast growth factor (bFGF or FGF2), orHepatocyte growth factor (HGF).
 8. A collagen material according toclaim 1, wherein a shape of the material is a ribbon, a sheet, a tube, asponge, a grain, a rod, a ring, a spiral, a spring, a disk, a dome or ablock.
 9. A collagen material according to claim 1, wherein the materialis designed so as to become almost equal to the orientation of each siteof a normal tissue from a living organism.
 10. A collagen materialaccording to claim 1, wherein the material contains oxygen.
 11. A methodof producing a collagen material, wherein the method comprises: a stepfor preparing a string collagen gel formed and orientated by collagensolution being extruded through a nozzle into a container containing aphosphate buffered saline (PBS), and giving a flow of a fixed directionto the collagen solution wherein a shape of the collagen gel is astring, and the direction of the orientation of the string collagen gelis a direction of the string shape, a step for obtaining multiple stringcollagen gels wherein the orientation are controlled, a step forarranging the multiple collagen gels in a desired shape, and a step fordrying the multiple collagen gels arranged in the desired shape tocouple with the collagen gels each other.
 12. A method of producing acollagen material according to claim 11, wherein the desired shape is aplanar shape and/or a three-dimensional shape.
 13. A method of producinga collagen material according to claim 11, wherein the desired shape isa ribbon, a sheet, a tube, a rod, a ring, a spiral, a disk, a dome or ablock.
 14. A method of producing a collagen material according to claim11, wherein the step for drying the collagen gels is carried out byfreeze dry.